Les biofilms bactériens : leur importance en santé animale et en santé publique

Bacterial biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that is attached to a surface. Biofilms protect and allow bacteria to survive and thrive in hostile environments. Bacteria within biofilms can withstand host immune responses, and are much less susceptible to antibiotics and disinfectants when compared to their planktonic counterparts. The ability to form biofilms is now considered an attribute of many microorganisms. Diseases associated with biofilms require novel methods for their prevention, diagnosis and treatment; this is largely due to the properties of biofilms. Furthermore, the presence of biofilms on surfaces found at farms, slaughterhouses or food processing plants will have an impact on the efficacy of disinfection protocols. Surprisingly, biofilm formation by bacterial pathogens of veterinary or zoonotic importance has received relatively little attention. The objective of this brief Review article is to bring awareness about the importance of biofilms to animal health stakeholders.(Translated by the authors)     

细菌排出泵、TolC家族和生物薄膜的作用机制研究进展

在多种重要病原体中存在抗生素排出泵,它与其它抗性一起构成了细菌显著的耐药性,其识别的底物非常广泛.G-菌中还存在与排出泵不同的另一种以蛋白质构成的排出系统,通过底物特异性内膜蛋白和外膜蛋白TolC家族的可逆性互作,也可以直接将包括小分子药物和大分子蛋白毒素等的多种分子从胞浆经周质区运到体外.粘附在内置医疗装置或组织中的细菌因其形成生物薄膜结构和以蛋白质、多糖为主的水性基质包裹的菌体也可以产生长期感染以及对抗生素的持久抗性,这种抗性与经典的质粒、转座子或突变产生的抗性不同.本文对细菌TolC家族和生物薄膜引起的抗性机制进行了综述.     

Biofilms and their relevance to veterinary medicine

Bacteria are renowned for their ability to tolerate and adapt to a wide range of adverse environmental conditions. The primary mechanism that facilitates these adaptations is thought to be the capacity to form and maintain biofilms. Within a biofilm, bacteria become attached to a surface where they exist in complex communities which are able to interact with each other through intracellular communication and thus rapidly adapt to changing environments. The organisms within biofilms are notorious for their resistance towards the host immune response and antibacterial agents compared to their free-living planktonic counterparts. Consequently, biofilms are of significant importance to both clinical and veterinary science. However, although bacterial infections are widely reported in animals their association with biofilms is rarely discussed. The aim of this review is to look at the characteristics of biofilm infections in humans and to relate this knowledge to veterinary science in order to assess their relevance in this area.     

细菌长极性菌毛的研究进展

长极性菌毛(long polar fimbriae,LPF)是一种最早发现于鼠伤寒沙门菌(Salmonella typhimurium,S.typhimurium)中的CU(chaperone/usher)类型的菌毛,后来该菌毛在其他许多细菌中也得到了鉴定。事实上,LPF不仅作为一种黏附素促进病原体黏附到易感细胞靶向部位,而且参与细菌生物被膜形成和感染组织嗜性。此外,LPF还能增强细菌在宿主细胞中的定植和持续存在能力,并介导宿主的炎症反应。在多种细菌,尤其是在对公共卫生有重要影响的肠出血性大肠杆菌(enterohemorrhagic Escherichia coli,EHEC)O157:H7菌株中,已经充分阐明了LPF是必不可少的毒力因子。现综述LPF的形态特征和分布、操纵子组成和功能、表达调节机制和生物学功能等方面的研究进展,旨在为研究LPF介导的细菌发病机制提供一定的理论基础。     

Microbiology of equine wounds and evidence of bacterial biofilms

Horse wounds have a high risk of becoming infected due to their environment. Infected wounds harbour diverse populations of microorganisms, however in some cases these microorganisms can be difficult to identify and fail to respond to antibiotic treatment, resulting in chronic non-healing wounds. In human wounds this has been attributed to the ability of bacteria to survive in a biofilm phenotypic state. Biofilms are known to delay wound healing, principally due to their recalcitrance towards antimicrobial therapies and components of the innate immune response. This study describes the presence of bacterial biofilms within equine wounds. Thirteen 8-mm diameter tissue samples were collected from (n=18) chronic wounds. Following histological staining, samples were observed for evidence of biofilms. Fifty one wounds and control skin sites were sampled using sterile swabs. Control skin sites were on the uninjured side of the horse at the same anatomical location as the wound. The isolated bacteria were cultured aerobically and anaerobically. The biofilm forming potential of all the isolated bacteria was determined using a standard crystal violet microtitre plate assay. Stained tissue samples provided evidence of biofilms within 61.5% (8 out of 13) equine wounds. In total 340 bacterial isolates were identified from all the equine wound and skin samples. Pseudomonas aeruginosa and Enterococcus faecium were the most predominantly isolated bacterial species from equine wound and skin samples respectively. Staphylococcus was the most commonly isolated genus in both environments. Bacteria cultured from chronic and acute wounds showed significantly (P<0.05) higher biofilm forming potential than bacteria isolated from skin. This paper highlights preliminary evidence supporting the presence of biofilms and a high microbial diversity in equine chronic wounds. The presence of biofilms in equine wounds partly explains the reluctance of many lower limb wounds to heal. Non-healing limb wounds in horses are a well documented welfare and economic concern. This knowledge can be used to shape future treatments in order to increase the healing rate and decrease the costs and suffering associate with equine wounds.     

Immune evasion by pathogens of bovine respiratory disease complex

Bovine respiratory tract disease is a multi-factorial disease complex involving several viruses and bacteria. Viruses that play prominent roles in causing the bovine respiratory disease complex include bovine herpesvirus-1, bovine respiratory syncytial virus, bovine viral diarrhea virus and parinfluenza-3 virus. Bacteria that play prominent roles in this disease complex are Mannheimia haemolytica and Mycoplasma bovis. Other bacteria that infect the bovine respiratory tract of cattle are Histophilus (Haemophilus) somni and Pasteurella multocida. Frequently, severe respiratory tract disease in cattle is associated with concurrent infections of these pathogens. Like other pathogens, the viral and bacterial pathogens of this disease complex have co-evolved with their hosts over millions of years. As much as the hosts have diversified and fine-tuned the components of their immune system, the pathogens have also evolved diverse and sophisticated strategies to evade the host immune responses. These pathogens have developed intricate mechanisms to thwart both the innate and adaptive arms of the immune responses of their hosts. This review presents an overview of the strategies by which the pathogens suppress host immune responses, as well as the strategies by which the pathogens modify themselves or their locations in the host to evade host immune responses. These immune evasion strategies likely contribute to the failure of currently-available vaccines to provide complete protection to cattle against these pathogens.     

Novel genes associated with biofilm formation of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is a gram-negative bacterium and is the causative agent of swine pleuropneumonia, a highly contagious respiratory disease. Biofilm formation is an important ability possessed by numerous bacterial pathogens. The purpose of this study was to identify and characterize biofilm mutants of A. pleuropneumoniae serotype 1 strain S4074 created using a mini Tn-10 transposon. The transposon library was screened to identify mutants with a modified ability to form biofilms in polystyrene microtiter plates. A total of 1200 mutants were screened and the analysis identified 24 mutants that exhibited abnormal biofilm formation, at least 16 unique genes were identified. Most genes identified in the enhanced-biofilm mutants encoded proteins with unknown functions, whereas most genes identified in the biofilm-reduced mutants encoded proteins related to transport, protein synthesis and nucleic acid synthesis. Approximately 50% of genes, including hns, potD2, ptsI, tig and rpmF, identified in our screen have been previously associated with biofilm formation in A. pleuropneumoniae and other bacterial species, and thus validated the screening method. The rest of genes identified, such as APL_0049, APL_0637 and APL_1572, have not been previously associated with biofilm formation. Interestingly, gene APL_0049 was previously seen among the genes differentially expressed during a natural infection of pig lungs. Preliminary characterization of the mutants was also initiated by assessing their hydrophobicity, their biofilm matrix composition and their ability to adhere to a polystyrene surface or NPTr cells. Based on the preliminary characterization, some of the mutants identified appear to have deficiencies during the initial attachment or growth of the biofilm. In conclusion, transposon mutagenesis analysis allowed the identification of new genes associated with biofilm formation in A. pleuropneumoniae.     

Public health and research perspectives on the microbial contamination of foods

Scientific advances in methodology and epidemiology have resulted in a renewed awareness of foodborne disease, and increased contact among nations of the world has stimulated rapid global distribution of foods as well as foodborne pathogens. New food vehicles are being identified for old, familiar pathogens, and new pathogens are being discovered. Current research in food microbiology has spurred development of rapid and specific methods to identify these pathogens and to assess their virulence. Organisms of recent interest, such as Bacillus, Yersinia, Campylobacter, Listeria, Sporothrix, Giardia, Cryptosporidium, and Anisakis, are the foci of new investigations, as are the more familiar foodborne pathogens, Salmonella, Shigella, Clostridium, Staphylococcus, Entamoeba and Ascaris. Some foodborne organisms, such as parasitic protozoa, serve as hosts for unique bacterial and viral symbionts but also might become infected with mammalian viruses. The remote possibility of the transmission of human immunodeficiency viruses in foodborne protozoa is discussed.     

Control of bacterial and fungal biofilms by natural products of Ziziphus joazeiro Mart. (Rhamnaceae)

The aim of this study is to verify the action of the aqueous leaf extract Ziziphus joazeiro in the eradication of bacterial and fungal biofilms, and to compare these effects with the stem bark extracts, as well as with conventional standard drugs. The presence of secondary metabolites was observed through phytochemical prospection assays. The effect of the aqueous extract on microbial biofilm formation was observed by OD600 nm absorbance and the crystal violet assay. For bacterial and fungal biofilms, chlorhexidine gluconate and fluconazole, respectively, were used as positive controls. Phytochemical characterization showed the presence of secondary metabolite classes common to both extracts such as flavonoids, steroids and saponins. In particular, in the aqueous leaf extract phenols, condensed tannins and alkaloids were observed. Eradication results using the aqueous leaf extract showed an inhibition of the microbial biofilm mass, moreover the biofilms were more sensitive to the bark extract, which presented a greater inhibition number and an action similar to standard drugs. It is important to highlight the leaf extract showed significant eradication at the lowest concentrations for mature yeast biofilms, thus demonstrating its potential to modify microbial resistance susceptibility. Bacterial and fungal biofilm eradication results using the Ziziphus joazeiro aqueous extracts presented a biofilm inhibition effect for both, moreover the results support the ethnopharmacological knowledge surrounding the use of Ziziphus joazeiro stems in the community. In comparison, the bark extract presented a more effective treatment than the leaf extract against biofilms, presenting inhibition levels similar to the used standard drugs.     

Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks

Ticks are known or suspected vectors for a wide range of bacterial pathogens. One of the first steps for tick-borne risk assessment is the detection of these pathogens in their vectors. In the present study, a broad-range PCR amplification of the eubacterial gene encoding the 16S rRNA gene combined with Temporal Temperature Gradient gel Electrophoresis (TTGE) was evaluated as a method allowing the one-step detection of bacterial pathogen DNA in ticks. Firstly, DNA extracts from bacteria known to be tick-borne pathogens, i.e., Borrelia burgdorferi lato sensu, Anaplasma phagocytophilum, Spotted Fever Group (SFG) Rickettsia spp., were used to establish a TTGE pathogen DNA reference marker. Secondly, we used broad-range PCR-TTGE to detect the presence of DNA from these three pathogens in 55 DNA extracts from pools of 10 nymphal Ixodes ricinus ticks, which have been previously shown to carry DNA from at least one of those bacteria by specific PCR. Among the 20 B. burgdorferi specific-PCR samples, 15 (75%) were also found to be positive using PCR-TTGE. Sixteen of the seventeen (94%) Rickettsia spp. PCR-specific samples were positive using PCR-TTGE detection and all PCR-specific positive extracts (11/11, 100%) for A. phagocytophilum were also positive using PCR-TTGE. Moreover, we identified unexpected bacterial sequences that were not related to any of the three pathogens such as a sequence related to Spiroplasma sp. Thus, broad-range PCR-TTGE allowed the single step detection of DNA from up to 3 pathogens in the same co-infected samples as well as detection of DNA from unexpected bacteria.     

The effect of lemongrass oil and its major components on clinical isolate mastitis pathogens and their mechanisms of action on Staphylococcus aureus DMST 4745

The aims of this study were to investigate the antibacterial activity of lemongrass oil (LG) and its major components which were citral, geraniol and myrcene, against four strains of clinically isolated bovine mastitis pathogens, including Staphylococcus aureus, Streptococcus agalactiae, Bacillus cereus and Escherichia coli by the broth microdilution method, as well as their activity on S. aureus biofilm formation. Attempts to clarify their mechanisms of action by investigation of the effects on intracellular material leakage and morphological changes of S. aureus DMST 4745 were also made. The results demonstrate that S. agalactiae and B. cereus are more susceptible to LG, citral and geraniol than S. aureus and E. coli. Moreover, they also inhibit S. aureus biofilm formation and exhibit effective killing activities on preformed biofilms. The LG appears to have multiple targets in the bacterial cell, depending on concentration used as well as the amount of its components.     

Susceptibility of bacterial pathogens against lincomycin/spectinomycin (1/2), penicillin G/neomycin (1/1), and penicillin G/dihydrostreptomycin (1/1) as determined in the BfT-GermVet monitoring program 2004-2006

A total of 368 bacterial pathogens, including 72 coagulase-positive and coagulase-variable staphylococci, 97 beta-haemolytic streptococci, 51 Escherichia coli, 75 Pasteurella multocida, 25 Mannheimia haemolytica, 25 Pseudomonas aeruginosa, and 23 Arcanobacterium pyogenes, were investigated for their susceptibility to the three combinations of antimicrobial agents lincomycin/spectinomycin (1/2), penicillin G/neomycin (1/1), and penicillin G/dihydrostreptomycin (1/1) in comparison to their susceptibility to the corresponding single substances. When comparing the minimum inhibitory concentrations (MICs) determined for any of the three combinations with those for the single substances, the lowest MIC of one of the two substances usually determined the MIC of the combination.This observation was made for all three combinations and all bacterial pathogens tested.Thus, it is assumed that the combination of lincomycin with spectinomycin as well as that of penicillin with either neomycin or dihydrostreptomycin resulted in an extended spectrum of target bacterial pathogens rather than in an increase in antimicrobial efficacy.     

The effect of lemongrass oil and its major components on clinical isolate mastitis pathogens and their mechanisms of action on Staphylococcus aureus DMST 4745

The aims of this study were to investigate the antibacterial activity of lemongrass oil (LG) and its major components which were citral, geraniol and myrcene, against four strains of clinically isolated bovine mastitis pathogens, including Staphylococcus aureus, Streptococcus agalactiae, Bacillus cereus and Escherichia coli by the broth microdilution method, as well as their activity on S. aureus biofilm formation. Attempts to clarify their mechanisms of action by investigation of the effects on intracellular material leakage and morphological changes of S. aureus DMST 4745 were also made. The results demonstrate that S. agalactiae and B. cereus are more susceptible to LG, citral and geraniol than S. aureus and E. coli. Moreover, they also inhibit S. aureus biofilm formation and exhibit effective killing activities on preformed biofilms. The LG appears to have multiple targets in the bacterial cell, depending on concentration used as well as the amount of its components.     

Comparative Gut Microflora,Metabolic Challenges,and Potential Opportunities

Bacteria colonize practically every habitat in the nature. Bacterial community of the gastrointestinal tract (GIT) is one of the major metabolic tissues of animals. Still, its species composition is not known, because methods that have been previously used for bacterial analysis do not capture the species present. The bacterial community within the GIT not only has intracommunity interactions, but it also interacts with the host tissues. For the host, it is essential to tolerate commensal bacteria and to recognize and fight intestinal pathogens. In addition to recognized pathogens, there are many functional bacterial groups, which produce metabolites considered harmful for the host. Such functional bacterial groups include protein-fermenting bacteria that produce toxic end products in their amino acid metabolism, which is why a high-protein diet is often considered unhealthy. Carbohydrates are preferred substrates for gastrointestinal bacteria, but in the distal colon, they may become depleted, and putrefaction becomes the dominating type of fermentation. This largely accounts for the health effects of slowly fermenting dietary fibers.Microbial communities in the GIT of different animals have developed hand-in-hand with their digestion strategy. In monogastric animals, bacterial fermentation has concentrated in the lower GIT, whereas in ruminants bacteria are responsible for the initial digestion of the diet. Rumen-like sites with intense bacterial fermentation are found in most animals, but no other group of animals totally depends on bacteria. The response of rumen bacteria to feed components can be evaluated by an in vitro rumen simulation, which measures relevant fermentation parameters, such as the yield of bacterial biomass (protein) and volatile fatty acids (energy). In monogastric animals, it is essential that the host has measures to prevent bacterial competition for substrates in regions in which absorption takes place; the use of prophylactic antibiotics for production animals has aided this.Availability of good methods for monitoring the total bacterial communities and their metabolism is a prerequisite for informative studies in the future.     

Putative probiotic Lactobacillus spp. from porcine gastrointestinal tract inhibit transmissible gastroenteritis coronavirus and enteric bacterial pathogens

A total of 310 bacterial strains isolated from the porcine gastrointestinal tract were tested for their activity against transmissible gastroenteritis (TGE) coronavirus and other enteric pathogens. Based on activity, the strains Probio-38 and Probio-37 were selected as potential probiotics and identified as Lactobacillus plantarum Probio-38 and Lactobacillus salivarius Probio-37 respectively by 16S rRNA gene sequencing. Supernatants of these strains inhibited TGE coronavirus in vitro in ST cells, without any cytopathic effect even after 72 h of incubation. Both the strains exhibited high survival in synthetic gastric juice. The strains were resistant to 5% porcine bile and exhibited antimicrobial activity against all the 13 enteric bacterial pathogens tested. These strains also exhibited resistance to most of the antibiotics analyzed. The inhibition of transmissible gastroenteritis coronavirus and enteric bacterial pathogens as well as the bile tolerance, high survival in gastric juice, and the antibiotic resistance indicate that the two isolated bacterial strains are ideal probiotic candidates for animal application after proper in vivo experiments.     

Investigation of aural bacterial and fungal flora following otitis in captive okapi (Okapia johnstoni)

Ten cases of otitis in nine captive okapi were observed at a captive zoological institution. Duration of clinical signs ranged from 3 days to 7 months. Two individuals each had a recurrence. A variety of fungi and bacteria were isolated from the aural canals of healthy okapi and their environments. Numerous different bacterial and fungal pathogens were cultured. Surveys investigating the occurrence of ear disease and related environmental factors from 15 other institutions that house okapi revealed otitis was only seen at one institution. While it appears that many bacterial and fungal pathogens are found in the okapi ear canal, it is suspected that environmental conditions, such as fungal load, air and hay quality, humidity, and temperature may be predisposing factors leading to ear infections in this species.     

Genetic fingerprinting (PFGE) of bacterial isolates for their molecular epidemiology

The determination of fragment patterns of bacterial genomic DNA after digestion with long-range-cutting restriction enzymes and their separation in pulsed-field-gelelectrophoresis is currently regarded as reliable fingerprinting tool for surveillance and identification of infectious sources and routes of bacterial pathogens. The PFGE based molecular fingerprinting has been applied and used for a broad range of bacterial species. As a prerequisite for its application in national and international network studies (German PulseNet, PulseNet USA, Salmgene) a common protocol for rapid production (1-2 days) of standardised and electronically readable fragment patterns has been developed. National and international outbreaks, diffuse outbreaks and long term surveillance of the ups and downs of pathogens have been successfully carried out by means of PFGE studies. The international outbreak of Salmonella enterica serovar Oranienburg (2001) due to contaminated chocolate is described here underlining the usefulness of PFGE as genetic fingerprinting for epidemiological purposes.     

Relevance in pathogenesis research

Research on pathogenesis of bacterial diseases involves exploration of the intricate and complex interactions among pathogen, host, and environment. Host-parasite-environment interactions that were relatively simple were the first to be understood. They include intoxications in which ingestion of a powerful bacterial toxin was sufficient to cause disease. In more complex cases bacteria occupy a variety of niches in the host and attack at an opportune time. Some bacterial pathogens have a brief encounter with the host; others are long-term guests. This variety of relationships involves a wide range of strategies for survival and transmission of bacterial pathogens. Molecular genetics, genomics and proteomics have facilitated understanding of the pathogens and hosts. Massive information often results from such studies and determining the relevance of the data is frequently a challenge. In vitro studies often attempt to simulate one or two critical aspects of the environment, such as temperature, pH, and iron concentration, that may provide clues as to what goes on in the host. These studies sometimes identify critical bacterial virulence factors but regulation of bacterial virulence and host response is complex and often not well understood. Pathogenesis is a process of continuous change in which timing and degree of gene expression are critical and are highly regulated by the environment. It is impossible to get the full picture without the use of natural or experimental infections, although experimental infections involve ethical and economic considerations which may act as a deterrent.     

细菌生物膜与细菌耐药性研究进展

细菌生物膜是一种包裹于细胞外多聚物基质中的黏附于非生物或生物表面的微生物菌落。作为一种生存策略,绝大多数细菌在合适的条件下都会产生生物膜,生物膜状态下的细菌相对其游离状态有着更强的耐药性,是导致临床上出现难治性感染的重要原因之一。主要综述了生物膜的形成、耐药机制及抗生物膜的策略,以便寻找有效控制生物膜相关感染的手段,指导临床合理用药和新药开发。     

羔羊痢疾的临床诊断及中西医治疗措施

羔羊痢疾是一种细菌性肠道疾病,此类疾病主要由大肠杆菌、魏氏梭菌、沙门氏菌、肠球菌引起。病菌通过口咽道进入动物体内,并随着粪便排泄出来,造成肠道炎症和病原体的大量排放,同时也会导致严重的腹泻和消化道不适。随着羊养殖规模的增加和养殖密度的增加,羔羊痢疾的发生频率逐渐上升,对其生命和健康造成严重损害,必须引起高度重视。